The long term goal of this grant is to elucidate the processes that ensure the faithful maintenance of eukaryotic chromosomes, using the yeast Saccharomyces cerevisiae as a model system. Telomeres, the physicalends of the chromosome, are essential for the stability and integrity of yeast chromosomes. Yeast chromosomes end in -300 bps of Ci-3A/TGi_3 DNA. Yeast telomeric DNA is normally synthesized by the reverse transcriptase telomerase, although recombination can maintain yeast telomeric DNA in cells lacking telomerase. The general goal of this funding period is to understand how telomere replication is regulated in Saccharomyces. The first and major aim focuses on two helicases, Pif Ip and RrmSp. These helicases are highly similar to each other and are members of a helicase sub-family that is conserved from yeast to humans. PifIp and RrmSp both influence telomeres but not in the same way. Rrm3p appears to act in a late step in telomere replication that is proposed to be important for generating a substrate for telomerase and hence promotes telomerase. Piflp appears to act downstream of Rrm3p, and its actions inhibit telomerase. Aim 1 describes a series of genetic, biochemical, and DNA structural studies to understandhow Piflp and Rrm3pregulate telomerase. Wild type and mutant recombinant Piflp and Rrm3p will be purified and used to determinesubstrate preferences. Both proteins will be tested for the effects on telomerase activity in vitro. In vivo analysisof the mutant alleles will determine if the helicase functions of Piflp and Rrm3p are responsible for their effects on telomere replication. Chromatin immuno-precipitation (ChIP) will determine if Piflp and/or Rrm3p are physically associated with telomeric DNA. Genetic approaches will identify genes that have overlapping functions with Rrm3p and to determine if lack of Rrm3p triggers a telomere-specific checkpoint. In vitro and in vivo approaches will determine if Piflp inhibits telomerase by nucleolytic degradation of its substrate. The second aim is to understandhow a very different type of telomerase regulator, a telomere structural protein, governs access of telomeres to telomerase. Rif Ip and Rif2p are telomere binding proteins that act synergistically to limit telomere lengthening.ChIP will be used to determine if Rif proteins regulate access of telomeric DNA to telomerase by cell cycle or telomere lengthdependent binding. The effects of Rif proteins on replication timing of telomeres and on telomerase-independent, recombinational telomere maintenance will also be determined. The third aim is to identify additional genes whose mutation or over- expression increases telomerase mediated healing of broken chromosomes. There is increasing evidence that telomere replication has effects on both aging and cancer. As yeast telomeric DNA and the proteins that govern its properties are functionally and/or structurally conserved from yeast to humans, understanding telomere regulation in yeast is likely to be relevant to genetic instability in humans.